CN109402023A - A kind of mould proof Bacillus strain and its application in foodstuff preservation - Google Patents
A kind of mould proof Bacillus strain and its application in foodstuff preservation Download PDFInfo
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- CN109402023A CN109402023A CN201811487520.4A CN201811487520A CN109402023A CN 109402023 A CN109402023 A CN 109402023A CN 201811487520 A CN201811487520 A CN 201811487520A CN 109402023 A CN109402023 A CN 109402023A
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 59
- 238000004321 preservation Methods 0.000 title claims description 14
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 50
- 235000009566 rice Nutrition 0.000 claims abstract description 50
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 31
- 238000003860 storage Methods 0.000 claims abstract description 21
- 235000013339 cereals Nutrition 0.000 claims abstract description 16
- 238000012216 screening Methods 0.000 claims abstract description 11
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 6
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims abstract description 3
- 241000209094 Oryza Species 0.000 claims description 49
- 230000001580 bacterial effect Effects 0.000 claims description 36
- 239000001963 growth medium Substances 0.000 claims description 33
- 241000228197 Aspergillus flavus Species 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 22
- 239000001965 potato dextrose agar Substances 0.000 claims description 16
- 238000000855 fermentation Methods 0.000 claims description 12
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- 241000894006 Bacteria Species 0.000 claims description 11
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- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B9/00—Preservation of edible seeds, e.g. cereals
- A23B9/16—Preserving with chemicals
- A23B9/24—Preserving with chemicals in the form of liquids or solids
- A23B9/26—Organic compounds; Microorganisms; Enzymes
- A23B9/28—Microorganisms; Enzymes; Antibiotics
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- C12N1/02—Separating microorganisms from their culture media
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/85—Food storage or conservation, e.g. cooling or drying
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Abstract
The invention discloses a kind of antimycotic Bacillus strain and its screenings and application.The strain classification is named as bacillus licheniformis (Bacillus licheniformis) BYJ7, is preserved in China General Microbiological culture presevation administrative center, deposit number CGMCC 16705.The present invention also provides a kind of methods for preparing the bacillus culture solution and its Volatile Metabolites.Bacillus licheniformis (Bacillus licheniformis) of the present invention and its Volatile Metabolites can play good inhibiting effect to the growth of spoilage organisms main in grain storage (especially rice), applied widely, application prospect is good, potentiality to be exploited is strong；And have the features such as energy-efficient, environmental-friendly, it can be applied to the preparation of grain mold proof agent.
Description
Technical field
The invention belongs to novel bacterial screening technique and microorganisms technical fields, in particular to one plant of antimycotic bacillus
Belong to bacterial strain and its mould proof application in foodstuff preservation.
Background technique
Rice is one of southern region of China staple food crop, however due to by peasant household's individual plantation, storage method, with
And the influence of the factors such as the Yangtze river basin and South China's high temperature and humidity weather, rice is in storage, transport and sales section
It is serious by mycotoxin pollution condition, easily go mouldy.
It is mainly studied both at home and abroad from the following aspects in preserving rice method: first is that research rice ageing
Mechanism delays rice moisture, protein, starch and fat etc. in storage to control the storage period that ageing improves rice
Variation；Second is that by killing the adult adhered on rice, worm's ovum, bacterium or destroying its growing environment, to inhibit biological life
Long breeding, the storage phase of Lai Yanchang rice；Third is that rice is isolated with worm's ovum, bacterium, it is allowed to lose source of nutrition to improve greatly
Rice storage quality (the progress grain engineering technology of Wu Yanli, Pan Liai preserving rice and storage technology, 2013,
10:59-62).Wherein, for killing or inhibiting pathogenic bacteria this aspect on rice, the measure mainly taken on the market at present is
Using antistaling agent or chemical fumigation.Although being for presently the most cost-effective method, due to dosage with chemical preservative
It is exceeded, using frequently etc. reasons, the environmental pollutions such as soil, water body are also got worse.Remain in the chemical sterilization in grain
Agent can further influence human health by food chain, bring certain food-safety problem.For these reasons, it finds and opens
It is a certainty for Agricultural Development that hair, which can replace the preserving rice method of chemical preservative and safety non-pollution,.
Biological mildew inhibitor partially replaces chemical preservative to be used in the food fresh keepings such as meat, fruits and vegetables and processing at present
In.But in general, at present for the fresh-keeping method for storing of the grains such as rice mainly pass through the environmental Kuznets Curves such as controlled atmosphere or with change
Fumigant processing is learned to control microorganism growth.Bacillus is that a kind of be widely present in nature Gram-positive is rod-shaped thin
Bacterium, it is characterised in that a variety of antibacterial substances and enzyme can be generated, there is stronger antisepsis, and be separately cultured easy, answer
It is wide with prospect.But currently, not there is the related report that bacillus Volatile Metabolites are applied to grain mold proof preservation field also
Road.
Summary of the invention
The object of the present invention is to provide one plant of antifungi Bacillus strain and its application method, Volatile Metabolites
Mildew-proof function can be played by controlled atmosphere during foodstuff preservation, be a kind of simple and easy, high-efficiency environment friendly microbiological treatment side
Method has a vast market foreground.
The invention discloses a kind of antifungi Bacillus strain, the classification naming of the bacterial strain is bacillus licheniformis
(Bacillus licheniformis) BYJ7, depositary institution: China General Microbiological culture presevation administrative center (CGMCC),
Deposit number: CGMCC No.16705, preservation date: on November 5th, 2018.
The invention also discloses a kind of screening technique of antifungi Bacillus strain, method particularly includes: select multi-region
Domain separates culture dish, and potato dextrose agar PDA is added in partition side, for cultivating Aspergillus flavus；The other side adds
Enter TSA or LB culture medium to cultivate bacillus.The activated bacillus of experimental group picking is applied on culture dish, coating
Region is consistent；Blank control group then without above-mentioned painting work, later with a small amount of Aspergillus flavus spore of bamboo stick picking with
Spot printing mode is seeded to the center of one side region of culture dish PDA culture medium；Inoculation is completed, after the sealing of sealed membrane bilayer, in
It is inverted culture one week in 25 DEG C of incubators, and every measurement each group Aspergillus flavus bacterial plaque diameter for 24 hours, and calculates bacteriostasis rate；It chooses
Bacteriostasis rate is greater than 15% bacterial strain, goes out the best antifungi Bacillus strain by index screening of bacteriostasis rate.The present invention
Screening technique have universality, the screening of all grain mold proof bacteriums can be generalized to, the fermentation liquid for the bacterium screened can
Volatile gas is discharged, the volatile gas is good to mould inhibitory effect.Bacillus licheniformis (Bacillus of the present invention
Licheniformis) BYJ7 is the bacterial strain that the produced volatile gas of one of which that this method is screened inhibits effect best.
The invention also discloses the fermentation liquid systems of bacillus licheniformis (Bacillus licheniformis) BYJ7 a kind of
Preparation Method, it is characterised in that the method are as follows: by the bacillus licheniformis (Bacillus licheniformis) after activation
BYJ7 is inoculated in the conical flask equipped with TSA or LB liquid medium with 2%-10% (v/v) inoculum concentration, 37 DEG C, 180rpm it is close
Overnight incubation is closed to get the fermentation of bacillus liquid containing volatility antibacterial metabolin is arrived.
The invention also discloses a kind of sides screened most suitable antifungi Bacillus strain growth and produce the culture medium of gas
Method.
Screening of Media method are as follows: select multizone to separate culture dish, wherein potato glucose fine jade is added in partition side
Rouge culture medium (PDA), for cultivating Aspergillus flavus；Culture medium (NB culture medium, TSA culture medium, TSB- to be screened is added in the other side
YE culture medium) to cultivate bacillus.Remaining processing step and Testing index and above-mentioned screening antifungi bacillus bacterium
Strain method is identical.
Preferably, the formula of the Bacillus strain culture medium TSA culture medium are as follows: 15g tryptone, 5g chlorination
Sodium, 5g soy peptone, 1.5% agar, 1L deionized water.
Bacillus licheniformis (Bacillus licheniformis) BYJ7 of the present invention can be applied to prepare grain
Fumigant or antistaling agent are stored, the growth and breeding of the moulds such as aspergillus flavus, Aspergillus ochraceus can be inhibited in foodstuff preservation.
The grain is preferably rice.
The present invention further discloses a kind of rice storage methods:
1) closing storage environment is cleaned and is sterilized, be put into the rice of dry cleansing；
2) using the fermentation liquid of the bacillus licheniformis (Bacillus licheniformis) BYJ7: dilution fermentation
Liquid, so that bacteria concentration is 106-109CFU/mL；
3) rice and the fermentation liquid after dilution in step 2) are fumigated in same confined space, the fermentation liquid after dilution
Volume ratio with grain storage space is 0.5%-1%；The relative humidity for keeping 60%-90%, is placed in preservation at 20-30 DEG C.
It simulates in rice storage experiment, by the sterile-processed rear inoculation Aspergillus flavus of rice, while utilizing lichens gemma bar
Bacterium (Bacillus licheniformis) BYJ7 Volatile Metabolites carry out suffocating treatment to it, observe the mouldy situation of rice
And total plate count in rice is measured according to national standard GB4789.2-2016.Research finds bacillus licheniformis (Bacillus
Licheniformis) BYJ7 Volatile Metabolites have obvious inhibiting effect to the total plate count in rice, and inhibiting rate can
Up to 38%.
By the above method, the present invention filters out one plant of microbial strains, passes through DNA gel QIAquick Gel Extraction Kit
(GE0101), pClone007Vector Kit kit (TSV-007) step and PCR method extraction purification and the bacterial strain is expanded
Genomic DNA, the sequence by measuring 16S rDNA carries out strain idenfication.By sequence measured by the strain in GenBank
BLAST comparative analysis is carried out in database, discovery and bacillus licheniformis (Bacillus in comparing the highest result of matching degree
Licheniformis) the 16S rDNA sequence homology highest of Pb-WC09001.It can determine that strain is bacillus licheniformis, life
Entitled Bacillus licheniformis BYJ7.The biological deposits information of the bacterial strain are as follows: China General Microbiological culture presevation
Administrative center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), culture presevation CGMCC No.16705.
Beneficial effects of the present invention:
Bacillus licheniformis (Bacillus licheniformis) BYJ7 and its Volatile Metabolites tool of the present invention
Standby certain antibacterial ability.According to simulation rice storage test as a result, bacillus licheniformis (Bacillus licheniformis)
BYJ7 Volatile Metabolites can restrain or delay the growth of the spoilage organisms such as Aspergillus flavus during foodstuff preservation, to rise
It is acted on to anti-mildew fresh-keeping, ensures stored grain safety.
Bacillus of the present invention resistance itself is strong, and antimicrobial spectrum is wide, can be applied to plurality of cereals storage environment
In, application prospect is good, potentiality to be exploited is strong.
Bacillus of the present invention is natural grain mold proof agent, can replace a large amount of chemical fumigant quilts in actual production
It uses, safety is had both to human body and environment.
Detailed description of the invention
Fig. 1 bacterial strain BYJ7 of the present invention is to aspergillus flavus inhibitory effect figure (culture 120h and 192h).Wherein left side is pair
According to group, right side is processing group.
Fig. 2 bacterial strain BYJ7 of the present invention is to aspergillus flavus fungistatic effect curve graph.Wherein abscissa is incubation time, indulges and sits
It is designated as bacteriostasis rate.
Fig. 3 bacterial strain BYJ7 of the present invention is under the conditions of different culture medium to aspergillus flavus fungistatic effect curve graph.It is wherein horizontal
Coordinate is incubation time, and ordinate is bacteriostasis rate.
Fig. 4 simulate volatile organic matter used in rice storage test (Volatile organic compounds,
VOCs bottle figure) is tested.
Fig. 5 simulates total plate count variation in rice storage test.Wherein abscissa is the time, and ordinate is the bacterium in rice
Fall sum.
The 16S rDNA sequence Neighbor-Joining phylogenetic tree of Fig. 6 bacterial strain BYJ7 of the present invention.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of that invention
In the case where essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.
Unless otherwise specified, in the embodiment of the present invention experimental material, reagent and instrument used etc. can by commercially available acquisition,
If not particularly pointing out, conventional means that technological means used in embodiment is well known to the skilled person.
Embodiment 1: Bacillus strain and its Volatile Metabolites of the present invention measure Aspergillus flavus fungistatic effect
And compared with other Bacillus strains
1. experimental method
Multizone is selected to separate culture dish, potato dextrose agar (PDA) is added in partition side, for cultivating
Aspergillus flavus；LB culture medium is added to cultivate bacillus in the other side.The activated bacillus of processing group picking is applied to
On culture dish, dispensing area is consistent；Blank control group is then without aforesaid operations, i.e., not on the region plating medium
Inoculation.One side region of culture dish PDA culture medium is seeded in a manner of spot printing with a small amount of Aspergillus flavus spore of bamboo stick picking later
Center.
Inoculation is completed, after the sealing of sealed membrane bilayer, culture one week is inverted in 25 DEG C of incubators, and each every measurement for 24 hours
Group Aspergillus flavus bacterial plaque diameter, and bacteriostasis rate is calculated as follows:
Bacteriostasis rate (%)=(blank control group bacterial plaque diameter-experimental group bacterial plaque diameter)/blank control group bacterial plaque diameter ×
100%
Potato dextrose agar (PDA): potato (purchased from local supermarket) peeling stripping and slicing weighs 200g, adds
Enter 1000mL deionized water, boil 30min, filtered through gauze is added glucose 20g, stirs evenly, add distilled water mend to
1000mL, the high pressure steam sterilization 20min at 121 DEG C.
LB culture medium prescription: 10g peptone, 10g sodium chloride, 5g yeast extract, 1.5%-2% agar, 1L deionized water, in
High pressure steam sterilization 20min at 121 DEG C.
It includes that bacterial strain of the present invention and other three bacillus bacterial strains, the above bacterial strain are equal that bacillus is used in experiment
It is provided by Zhejiang University's biosystem engineering and Food Science institute laboratory.
2. experimental result and analysis
As shown in table 1, Fig. 2 is bacterial strain of the present invention and its Volatile Metabolites to Huang to experimental data (bacteriostasis rate)
Aspergillus fungistatic effect.Compared with other a few bacillus bacterial strains, bacterial strain of the present invention can reach higher to Aspergillus flavus
Bacteriostasis rate, and bacteriostasis rate is more stable.Bacillus strain and its Volatile Metabolites of the present invention are antibacterial to aspergillus flavus
It works well and more prominent.
Bacteriostasis rate (%) of the 1 four kinds of Bacillus strains of table to aspergillus flavus
Embodiment 2: Bacillus strain optimum medium screening of the present invention
1. experimental method
Multizone is selected to separate culture dish in experiment, wherein potato dextrose agar is added in partition side
(PDA), for cultivating Aspergillus flavus；Culture medium culture bacillus of the present invention to be screened is added in side.Processing group is chosen
Activated bacillus is taken to be applied on culture dish, coating range is the whole region of culture dish partition side；Blank pair
According to group then without aforesaid operations, i.e., it is not inoculated on the demifacet plate.The a small amount of Aspergillus flavus spore of picking is later with spot printing side
Formula is seeded to the center of one side region of culture dish PDA culture medium.
In experiment to cultivate bacillus of the present invention culture medium to be screened be NB culture medium, TSA culture medium,
Three kinds of TSB-YE culture medium.
NB culture medium prescription: 10g peptone, 3g beef extract, 5g sodium chloride, 1.5%-2% agar, 1L deionized water；It is high
Pressure steam sterilizing condition: 121 DEG C, 20min.
TSA culture medium prescription: 15g tryptone, 5g sodium chloride, 5g soy peptone, 1.5% agar, 1L deionized water；
High pressure steam sterilization condition: 121 DEG C, 20min.
TSB-YE culture medium prescription: 15g tryptone, 5g sodium chloride, 5g soy peptone, 1.5%-2% agar, 6.5g
Yeast extract, 1L deionized water；High pressure steam sterilization condition: 121 DEG C, 20min.
After inoculation is completed, sealed membrane seals two layers, culture is inverted in 25 DEG C of incubators, and yellow every measurement each group for 24 hours
Aspergillus bacterial plaque diameter, and bacteriostasis rate is calculated as follows:
Bacteriostasis rate (%)=(blank control group bacterial plaque diameter-experimental group bacterial plaque diameter)/blank control group bacterial plaque diameter ×
100%
2. experimental result and analysis
Fig. 3 is that bacterial strain of the present invention its Volatile Metabolites under different culture medium culture inhibit to imitate to Aspergillus flavus
Fruit.In three kinds of different culture mediums, bacillus of the present invention is on TSA culture medium to aspergillus flavus bacteriostasis rate and another the two phase
Than being substantially higher, 30% or so can achieve；And fungistatic effect is seen on the whole simultaneously with NB and two kinds of culture medium cultures of TSB-YE
It does not protrude, also without too big difference.Its reason may be due in TSA culture medium nitrogen source than both another sufficient and abundance,
It can play the role of that bacillus metabolism is promoted to generate Volatile Metabolites to a certain extent, to enhance it to yellow bent
The fungistatic effect of mould.It is the preferred of culture bacillus of the present invention that TSA culture medium, which can be obtained, by this experiment.
Embodiment 3: simulation rice storage test
1. rice experiment in vivo pre-processes
1.1 experimental materials, reagent and instrument
Experimental material: bacillus that northeast rice (purchased from local supermarket), aspergillus flavus, number are BYJ7, physiology salt
Water, sterile water, centrifuge tube, bamboo stick, plastic casing etc..
Experiment reagent: 0.3% liquor natrii hypochloritis, 0.1% liquor natrii hypochloritis, TSA fluid nutrient medium.
Laboratory apparatus: VOCs test bottle, shaking table, optical microscopy, baking oven, super-clean bench etc..
1.2 experimental method
(1) VOCs tests building for bottle
Nozzle filtering flask and a two tunnels straight trip piston are connected by rubber tube on two 250mL.Nozzle on one 250mL
Filtering flask seals with rubber stopper (bottle is used as culture bacillus), and nozzle filtering flask is sealed with sterile culture on another 250mL
Film sealing (places the rice by mould contamination) in this bottle.The device needs to carry out autoclave sterilization after putting up.
(2) prepared by bacillus bacteria suspension
A small amount of bacillus BYJ7 is scraped on plate is inoculated into the 250mL conical flask for filling 50mL liquid TSA culture medium
In, it is put into 37 DEG C of shaking tables and is incubated overnight.1mL is taken to be added to VOCs test bottle in bacillus bacteria suspension after being incubated overnight
One of them of device fills in the upper nozzle filtering flask of 50mLTSA fluid nutrient medium, and VOCs test bottle is put into shaking table
In cultivate 8h or so at 37 DEG C and reach its logarithmic phase and carry out experiment again with (the liquid TSA culture medium of control group is not inoculated with gemma
Bacillus bacteria suspension).
(3) prepared by mycotic spore suspension
Scrape aspergillus flavus mycotic spore on a small quantity in advance plus in the 5mL centrifuge tube of sterile water, oscillation mixes, under microscope into
Row spore count.Obtained bacterial concentration is diluted to 10 again3Cfu/mL, it is spare in 10mL centrifuge tube.
(4) rice pre-processes
Plastic casing 10min first is impregnated with 0.3% liquor natrii hypochloritis, outwells drying.It is put into rice in plastic casing, uses
0.1% liquor natrii hypochloritis did not had rice surface to impregnate 30s.It outwells and plastic casing is wrapped into preservative film after solution, be placed in baking oven
Interior 15min makes rice surface moisture up to closely dry state, takes out plastic casing.
(5) rice is placed
The above-mentioned processed rice of 100g is weighed to be added on another 250mL in nozzle filtering flask, after being all added,
The above-mentioned aspergillus spore suspension (10 of 200 μ L is added in the bottle for filling rice to every in super-clean bench3cfu/mL).It is use up when addition
Amount makes spore suspension be evenly distributed in rice.
(6) it cultivates
After sample addition finishes and seals device, it is put into culture (temperature: 29 DEG C in shaking table；Revolving speed: 150rpm).Divide later
The measurement of total number of molds in the 4th day (Day 4) and the 8th day (Day 8) rice sample progress rice is not taken.
2. influence of the bacillus Volatile Metabolites to total plate count in rice
2.1 experimental method
(1) it weighs 5g sample to set in the conical flask for filling 45mL physiological saline, acutely vibrates 5min, the sample of 1:10 is made
Even liquid.
(2) the even 400 μ L of liquid of 1:10 sample is drawn, is slowly infused in the sterile centrifugation tube for filling 3.6mL dilution along tube wall,
It mixes, the even liquid of sample of 1:100 is made.
(3) the even liquid of each 10 times of series of diluted samples of aforesaid operations system is pressed.
(4) according to the estimation to sample pollution situation, the even liquid of sample of 2~3 acceptable diluent degree is selected, draws 200 μ
For the even liquid of L sample in sterilized petri dishes, each dilution makees two plates.Meanwhile 200 μ L solution dilution blanks are drawn respectively and are added two
Make blank control in a sterilized petri dishes.
(5) 15mL~20mL PDA culture medium for being cooled to 46 DEG C (can be placed in 46 DEG C of ± 1 DEG C of constant water bath box in time
Middle heat preservation) pour plate, and rotating plate is uniformly mixed it.
(6) it is counted after cultivating 36-48h.
2.2 experimental result and analysis
Influence of the bacillus Volatile Metabolites to total plate count in rice is as shown in Figure 5.
As shown in figure 5, take above-mentioned culture respectively the 4th day and the 8th day rice carry out total plate count measurement.The 4th
It when, the total plate count in control group and processing group is respectively 5.2 × 103Cfu/g and 3.2 × 103Cfu/g, bacteriostasis rate are reachable
38%；At the 8th day, the total plate count in control group and experimental group was respectively 2 × 105Cfu/g and 1.5 × 105Cfu/g, suppression
Bacterium rate is 25%.Illustrate that bacillus Volatile Metabolites have certain inhibiting effect to rice mildew, in grain storage is mould proof
There is certain application prospect.
Embodiment 4: the Species estimation of Bacillus strain of the present invention
1. experimental method
The total DNA of bacterial strain of the present invention is extracted according to pClone007Vector Kit kit (TSV-007) step,
The 16S rDNA gene of the bacterium is expanded with primer M13F-47 and M13R-48, is sequenced after recycling amplified production, passes through measurement
The sequence of 16S rDNA carries out strain idenfication.The sequence results of acquisition carry out Blast comparison in NCBI, from the number of GenBank
According to acquisition in library and bacterial strain 16S rDNA homologous recognised standard sequence data, simultaneously using MEGA software sequence of calculation similitude
Phylogenetic Analysis is made using the ortho position algorithm (Neighbor-Joining) that is connected.
2. result of implementation is analyzed
Bacterial strain of the present invention identifies that sequence results are as shown in SEQ ID NO.1 through 16S rDNA complete sequence determination.
The 16S rDNA of the bacterial strain is compared in NCBI using Blast, is the discovery that bacillus (Bacillus sp.) simultaneously
With its similar strain 16S rDNA sequence construct 16S rDNA phylogenetic tree as shown in fig. 6, and bacillus licheniformis
The homology highest of (Bacillus licheniformis) Pb-WC09001, it may be determined that bacterial strain of the present invention is lichens gemma
Bacillus (Bacillus licheniformis), and it is named as Bacillus licheniformis BYJ7.
The above description of the embodiment is only used to help understand the method for the present invention and its core ideas.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Appropriate to change and modify, these change and modification is also fallen within the protection scope of the claims of the present invention.
Sequence table
<110>Zhejiang University
<120>a kind of mould proof Bacillus strain and its application in foodstuff preservation
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1556
<212> DNA
<213>bacillus licheniformis (Bacillus licheniformis)
<400> 1
tgaggctcgc tgcaatcgcg tgtcgccctt agagtttgat cctggctcag gacgaacgct 60
ggcggcgtgc ctaatacatg caagtcgagc ggatagatgg gagcttgctc cctgatgtca 120
gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga 180
aaccggggct aataccggat gcttgattga accgcatggt tcaattataa aaggtggctt 240
cggctatcac ttacagatgg acccgcggcg cattagctag ttggtgaggt aacggctcac 300
caaggcgacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg 360
gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg 420
gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa aactctgttg ttagggaaga 480
acaagtaccg ttcgaatagg gcggcacctt gacggtacct aaccagaaag ccacggctaa 540
ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg 600
taaagcgcgc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc aaccggggag 660
ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc acgtgtagcg 720
gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct ggtctgtaac 780
tgacgctgag gcgcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc 840
cgtaaacgat gagtgctaag tgttagaggg tttccgccct ttagtgctgc agcaaacgca 900
ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg 960
cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 1020
cttgacatcc tctgacaacc ctagagatag ggcttcccct tcgggggcag agtgacaggt 1080
ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1140
aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa 1200
accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1260
gtgctacaat gggcagaaca aagggcagcg aagccgcgag gctaagccaa tcccacaaat 1320
ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa 1380
tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1440
ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct tttggagcca gccgccgaag 1500
gtgggacaga tgattggggt gaagtcgtaa caaggtaacc aagggcgaca cgcgat 1556
Claims (7)
1. a kind of antifungi Bacillus strain, it is characterised in that the classification naming of the bacterial strain is bacillus licheniformis
(Bacillus licheniformis) BYJ7, depositary institution: China General Microbiological culture presevation administrative center (CGMCC),
Deposit number: CGMCC No.16705, the deposit date is on November 5th, 2018.
2. a kind of screening technique of antifungi Bacillus strain, it is characterised in that the method are as follows: multizone is selected to separate
Culture dish, potato dextrose agar PDA is added in partition side, for cultivating Aspergillus flavus；TSA or LB is added in side
Culture medium is to cultivate bacillus；The activated bacillus of experimental group picking is applied on culture dish, and dispensing area is kept
Unanimously；Blank control group is then without above-mentioned painting work, later with a small amount of Aspergillus flavus spore of bamboo stick picking in a manner of spot printing
It is seeded to the center of one side region of culture dish PDA culture medium；Inoculation is completed, after the sealing of sealed membrane bilayer, is cultivated in 25 DEG C
It is inverted culture one week in case, and every measurement each group Aspergillus flavus bacterial plaque diameter for 24 hours, and calculates bacteriostasis rate；It is big to choose bacteriostasis rate
In 15% bacterial strain, go out the best antifungi Bacillus strain by index screening of bacteriostasis rate.
3. the zymotic fluid preparation method of antifungi bacillus described in a kind of claim 1, it is characterised in that the method are as follows: will
Bacillus licheniformis (Bacillus licheniformis) BYJ7 after activation is inoculated in dress by 2%-10% (v/v) inoculum concentration
Have in TSA or the conical flask of LB liquid medium, 37 DEG C, the closed overnight incubation of 180rpm to get to contain volatility antibacterial generation
Thank to the fermentation of bacillus liquid of object.
4. antifungi Bacillus strain described in claim 1 is preparing the application in foodstuff preservation fumigant or antistaling agent.
5. the application that antifungi Bacillus strain described in claim 1 inhibits aspergillus flavus, Aspergillus ochraceus in foodstuff preservation.
6. application as claimed in claim 5, it is characterised in that the grain is preferably rice.
7. a kind of rice storage method, it is characterised in that method is as follows:
1) closing storage environment is cleaned and is sterilized, be put into the rice of dry cleansing；
2) fermentation liquid prepared using claim 3 the method dilutes fermentation liquid, so that bacteria concentration is 106-109CFU/mL；
3) rice and the fermentation liquid after dilution in step 2) are fumigated in same confined space, the fermentation liquid and storage after dilution
The volume ratio in grain space is 0.5%-1%；The relative humidity for keeping 60%-90%, is placed in preservation at 20-30 DEG C.
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| CN110964668A (en) * | 2019-12-26 | 2020-04-07 | 浙江大学 | Anti-escherichia coli bacillus alvei strain and antiseptic application thereof in cosmetics |
| CN111690566A (en) * | 2020-07-03 | 2020-09-22 | 山东第一医科大学(山东省医学科学院) | Bacillus tequilensis and application thereof |
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| CN101457212A (en) * | 2008-12-10 | 2009-06-17 | 中国农业大学 | Screening, culturing and application of bacterial strain for simultaneous degrading aspergillus flavus toxin B1 and ochratoxin A |
| KR20140012411A (en) * | 2012-07-20 | 2014-02-03 | 순창군 | Bacillus licheniformis strain sck b11 having antimicrobial activity against pathogenic microorganism of soy sauce and degrading activity of biogenic amine and uses thereof |
| CN106381277A (en) * | 2016-08-31 | 2017-02-08 | 北京科技大学 | Method for removing aflatoxin B1 through using Bacillus licheniformis enzyme preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457212A (en) * | 2008-12-10 | 2009-06-17 | 中国农业大学 | Screening, culturing and application of bacterial strain for simultaneous degrading aspergillus flavus toxin B1 and ochratoxin A |
| KR20140012411A (en) * | 2012-07-20 | 2014-02-03 | 순창군 | Bacillus licheniformis strain sck b11 having antimicrobial activity against pathogenic microorganism of soy sauce and degrading activity of biogenic amine and uses thereof |
| CN106381277A (en) * | 2016-08-31 | 2017-02-08 | 北京科技大学 | Method for removing aflatoxin B1 through using Bacillus licheniformis enzyme preparation |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964668A (en) * | 2019-12-26 | 2020-04-07 | 浙江大学 | Anti-escherichia coli bacillus alvei strain and antiseptic application thereof in cosmetics |
| CN110964668B (en) * | 2019-12-26 | 2021-05-04 | 浙江大学 | Anti-escherichia coli bacillus alvei strain and antiseptic application thereof in cosmetics |
| CN111690566A (en) * | 2020-07-03 | 2020-09-22 | 山东第一医科大学(山东省医学科学院) | Bacillus tequilensis and application thereof |
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